Immunophenotyping of Human Regulatory T Cells

Regulatory T cells, also known as Tregs, play a pivotal role in maintaining homeostasis of the immune system and self-tolerance. Tregs express CD3, CD4, CD25, and FOXP3 but lack CD127. CD4 and CD3 identify helper T lymphocytes, of which Tregs are a subset. CD25 is IL-2Rα, an essential activation marker that is expressed in high levels on Tregs. FOXP3 is the canonical transcription factor, important in the development, maintenance, and identification of Tregs. CD127 is IL-7 receptor, expressed inversely with suppression, and is therefore downregulated on Tregs. Flow cytometry is a powerful tool that is capable of simultaneously measuring Tregs along with several markers associated with subpopulations of Tregs, activation, maturation, proliferation, and surrogates of functional suppression. This chapter describes a multicolor flow cytometry-based approach to measure human Tregs, including details for surface staining, fixation/permeabilization, intracellular/intranuclear staining, acquisition of samples on a flow cytometer, plus analysis and interpretation of resulting FCS files.